Compositions and methods for analyzing histidine phosphorylation

Abstract

A peptide is disclosed of the general structure: Z—W—Y, wherein Z and Y are independently a one to eight amino acid sequence wherein the amino acids are selected from glycine and alanine and W is a non-hydrolyzable pHis analogue. Such peptides can be used to produce sequence-independent anti-phosphohistidine antibodies. Also provided are antibodies that specifically bind to a peptide comprising a phosphohistidine (or a non-hydrolyzable pHis analogue) but fail to specifically bind to an identical peptide containing histidine instead of phosphohistidine.

1 . A peptide comprising the structure
   Z—W—Y
   wherein   Z is a sequence selected from the group consisting of X 1 , X 1 X 2 , X 1 X 2 X 3 , X 1 X 2 X 3 X 4  (SEQ ID NO: 1), X 1 X 2 X 3 X 4 X 5  (SEQ ID NO: 2), X 1 X 2 X 3 X 4 X 5 X 6  (SEQ ID NO: 3), X 1 X 2 X 3 X 4 X 5 X 6 X 7  (SEQ ID NO: 4) and X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8  (SEQ ID NO: 5);   
       W is an amino acid selected from 
       
         
           
           
               
               
           
         
         Y is a sequence selected from the group consisting of X 11 , X 11 X 12 , X 11 X 12 X 13 , X 11 X 12 X 13 X 14  (SEQ ID NO: 6), X 11 X 12 X 13 X 14 X 15  (SEQ ID NO: 7), X 11 X 12 X 13 X 14 X 15 X 16  (SEQ ID NO: 8), X 11 X 12 X 13 X 14 X 15 X 16 X 17  (SEQ ID NO: 9) and X 1 X 12 X 13 X 14 X 15 X 16 X 17 X 18  (SEQ ID NO: 10); wherein 
         X 1 , X 2 , X 3 , X 4 , Xs, X 6 , X 7 , X 8 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17  and X 18  are independently alanine or glycine. 
       
     
     
         2 . The peptide of  claim 1  further comprising a single cysteine, tyrosine or lysine linked to the amino or carboxy terminus of said peptide. 
     
     
         3 . The peptide of  claim 1  further comprising an N-terminal cysteine. 
     
     
         4 . The peptide of  claim 3  wherein the N-terminal amine is acylated and the C-terminal carboxylic acid group is replaced with an amide. 
     
     
         5 . The peptide of  claim 4  wherein both Z and Y are four amino acids in length. 
     
     
         6 - 10 . (canceled) 
     
     
         11 . A composition/library comprising a plurality of different peptides of  claim 2 . 
     
     
         12 . The composition of  claim 11  comprising 256 different peptides wherein each of said 256 peptides comprises the structure Cys-Z—W—Y. 
     
     
         13 . The composition of  claim 11  wherein each of said peptides comprise an N-terminal cysteine, said peptides being covalently coupled to a carrier protein through the side chain of said cysteine amino acid, said composition further comprising an adjuvant, wherein said peptides are emulsified in said adjuvant. 
     
     
         14 - 33 . (canceled)